The present invention relates generally to the fields of plants, such as maize, and to improved methods of plant breeding. More particularly, it provides methods of increasing yield in plants by introducing a gene encoding phosphinothricin acetyltransferase. The increased yield phenotype may be transferred to other lines of plants by crossing.
Ever since the human species emerged from the hunting-gathering phase of its existence, and entered an agricultural phase, a major goal of human ingenuity and invention has been to improve crop yield and to alter and improve the characteristics of plants. In particular, man has sought to alter the characteristics of plants to make them more tasty and/or nutritious, to produce increased crop yield or render plants more adaptable to specific environments.
Up until recent times, crop arid plant improvements depended on selective breeding of plants with desirable characteristics. Initial breeding success was probably accidental, resulting from observation of a plant with desirable characteristics, and use of that plant to propagate the next generation. However, because such plants had within them heterogenous genetic complements, it was unlikely that progeny identical to the parent(s) with the desirable traits would emerge. Nonetheless, advances in controlled breeding have resulted from both increasing knowledge of the mechanisms operative in hereditary transmission, and by empirical observations of results of making various parental plant crosses.
Attempts to improve commercially important traits in plants, for example, grain yield in corn and wheat, have consumed the energies of commercial plant breeders in the twentieth century. Clever and sophisticated breeding schemes have been devised, yet the rate of improvement of economically important characters has been only a few to several percent of the mean per year for the past several decades. For various crop plants, it has been established that roughly half of this improvement is due to improved husbandry practices, i.e., environmental effects rather than genetic changes effected by selection. (Lande and Thompson, 1990).
The record available from the crude crop breeding programs of the late nineteenth century through the present is littered with dead endsxe2x80x94failures, for one reason or another. For example, data on the ineffectiveness of mass selection for several corn ear characters as presented by Williams and Welton in 1915 are reproduced and discussed by Sprague and Eberhart (1977). Selection for long and short ears was not effective in separating the population into two distinct subpopulations defined by ear length. Yield, one of the most commercially valuable traits, has been the least responsive to selective breeding programs. Selection from 1907-1914 had no overall effect on yield. An examination of data on corn yield trials published by study stations in Illinois from 1860 to 1900 shows that many corn varieties were included for short test periods, then discarded because of poor yielding ability. (Sprague and Eberhart, 1977). This article refers to a report that visual selection practiced during inbreeding had little, if any, direct influence on yield in hybrid combinations. However, selection was effective for some other traits, e.g., maturity. Recurrent selection was somewhat more effective in improving breeding populations.
These failures to substantially alter plant characteristics are costly. Even the successes with recurrent selection may generally be described as incremental and long range improvements rather than mercurial saltatory jumps. Divergence of corn varieties for oil and protein content of grain was achieved if results over the 70 year history of a long-term study in Illinois are considered. However, improvement in yield has been less dramatic. Over the past 60 years, increases in yield due to genetic improvement have averaged only about one bushel/acre/year (Hallauer et al., 1988). Only a small population of hybrid plants produced commercially ever show enough improvement to be worth marketing. World-wide needs for plant derived food, both for animals and humans, warrant improved strategies. Plants are also finding uses in non-food products necessitating increased production. New methods are necessary for more efficient and successful plant breeding programs than are currently available.
Recent advances in molecular biology have expanded man""s ability to manipulate the germplasm of animals and plants. Genes controlling specific phenotypes, for example specific polypeptides that lend antibiotic or herbicide resistance, have been located within certain germplasm and isolated from it. Even more important has been the ability to take the genes which have been isolated from one organism and to introduce them into another organism. This transformation may be accomplished even where the recipient organism is from a different phylum, genus or species from that which donated the gene (heterologous transformation).
Attempts have been made to genetically engineer desired traits into plant genomes by introduction of exogenous genes. These techniques have been successfully applied in some plant systems, principally in dicotyledonous species. The uptake of new DNA by recipient plant cells has been accomplished by various means, including Agrobacterium infection (Nester et al., 1984), polyethylene glycol (PEG)-mediated DNA uptake (Lorz et al., 1985), electroporation of protoplasts (Fromm et al., 1986) and microprojectile bombardment (Klein et al., 1987). Unfortunately, the introduction of exogenous DNA into monocotyledonous species and subsequent regeneration of transformed plants has proven much more difficult than transformation and regeneration in dicotyledonous plants. However, techniques are now available for transformation of barley (Wan and Lemaux, 1994), wheat (Weeks et al., 1993), corn (Gordon-Kamm et al., 1990), and sorghum (Casas et al., 1993). The availability of these transformation techniques suggests that it may now be possible to address improvement of agronomic performance of a crop plant through the techniques of genetic engineering.
With the advent of genetic engineering techniques that are new opportunities for increasing yield in crops. One of the goals of this technology is to introduce genes into a crop that will increase yield and/or stabilize yield across multiple environments. However, no genetic elements that contribute to important agronomic characteristics, such as yield, have been identified and introduced into crops using the techniques of genetic engineering.
The enzyme phosphinothricin acetyl transferase (PAT) inactivates the active ingredient in the herbicides bialaphos or phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami et al., 1986; Twell et al., 1989) causing rapid accumulation of ammonia and cell death. Genes that encode the enzyme phosphinothricin acetyltransferase are obtainable from species of Streptomyces (e.g., ATCC No. 21,705) and include the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. These organisms synthesize numerous unique compounds, secondary metabolites, that often possess antibacterial, antitumor, or antiparasitic activity (Demain et al., 1983). Streptomyces viridochromogenes produces a broad spectrum tripeptide antibiotic phosphinothricyl-alanyl-alanine (phosphinothricin) [2-amino-4-(methylphosphinyl)-butanoic acid] (Bayer et al., 1972). The gene that encodes for phosphinothricin resistance has been designated pat and was first isolated from S. Viridochromogenes and shares extensive nucleotide sequence homology with the bar gene of S. hygroscopicus (Murakami et al., 1986; Thompson et al., 1987). The bar gene has been well studied and serves as a model to explain the mode of action of the pat gene. The bar gene encodes a phosphinothricin acetyltransferase, which acetylates the free NH2 group of phosphinothricin and thereby prevents autotoxicity in the producing organism (Murakami et al., 1986). Hence, the bar gene can be cloned to obtain resistance to the antibiotic bialaphos and can be used as a dominant selectable marker.
The bar gene has been cloned and used as a selectable marker in E. coli (Thompson et al., 1987) which is bialaphos sensitive (Murakami et al., 1986). The bar gene has also been expressed in tobacco, tomato, and potato plants (de Block et al., 1987; Leemans et al., 1987). The transgenic tobacco, tomato, and potato plants were completely resistant to high doses of the commercial formulations of both phosphinothricin and bialaphos (de Block et al., 1987). S. hygroscopicus is used for the commercial production of bialaphos (HerbiaceR, Meiji Seika Kaisha, Ltd., Yokohama, Japan) and is being used in agriculture as a nonselective herbicide (Thompson et al., 1987). Phosphinothricin is chemically synthesized and sold under the tradename of BastaR, Hoechst AG, Germany). Selection for bialaphos resistance can be made in the field by spraying seedlings with a 1% solution of Basta containing 200 g/L glufosinate (the ammonium salt of phosphinothricin).
The enzyme encoded by the bar gene is also involved in the bialaphos biosynthetic pathway in S. hygroscopicus. In addition to acetylation of phosphinothricin, the enzyme catalyzes acetylation of demethyl-phosphinothricin which is an intermediate in the biosynthetic pathway (Thompson et al., 1987). PAT, however, has no activity on any substrates other than phosphinothricin and demethyl-phosphinothricin, including no activity on the amino acid glutamate of which phosphinothricine is an analog (Thompson et al., 1987). Introduction of a gene encoding PAT into a plant would, therefore, be expected to confer resistance to the herbicide phosphinothricin on the plant, but not effect the phenotype of the plant in any other way.
The present invention relates to the surprising discovery that the introduction of a PAT gene into plants, such as corn, correlates with increased yield in the resultant plants. The invention therefore provides new methods and compositions for use in plant breeding and genetic engineering to specifically alter yield.
A transformed plant or transformed plant line is defined herein as a plant or plant line into which a DNA sequence, preferably from a source with which the recipient plant is not sexually compatible, has been introduced through the use of genetic engineering techniques.
A DNA sequence that encodes the enzyme phosphinothricin acetyl transferase (PAT) is herein defined as a xe2x80x9cPAT genexe2x80x9d, regardless of the source of the gene. Genes encoding PAT include the bar gene isolated from Streptomyces hygroscopicus and the pat gene isolated from Streptomyces viridochromogenes. 
pat gene compositions are described in U.S. Pat. No. 5,273,894, incorporated herein by reference, which patent also describes the use of the pat gene as a resistance marker. The pat gene is further exemplified by DNA segments that include a DNA sequence as set forth herein by SEQ ID NO:1 and that encode a polypeptide that includes an amino acid sequence as set forth in SEQ ID NO:2. A pat gene sequence with modified codon usage for advantageous expression in plants is described in U.S. Pat. No. 5,276,268, incorporated herein by reference. In U.S. Pat. No. 5,276,268, the DNA sequence termed sequence III, as shown in Table III, is the sequence adapted for expression in plants. This sequence is contemplated for use in the present invention.
The bar gene is currently the preferred gene for use with the present invention. The bar gene is described in issued European Patents EP 0,242,236 and EP 0,242,246, and shown in FIG. 2 thereof. EP 0,242,246 also discloses the pat gene in FIG. 9. The bar gene is further exemplified by DNA segments that include a DNA sequence as set forth herein by SEQ ID NO:3 and that encode a polypeptide that includes an amino acid sequence as set forth in SEQ ID NO:4.
Irrespective of the source of the gene, it is preferred that the PAT gene be operatively linked to a constitutive promoter so that the promoter functions to express the gene. In a currently preferred embodiment, the gene encoding PAT is constitutively expressed by functionally linking the gene downstream of a Cauliflower Mosaic Virus (CMV) 35S promoter. A variety of other promoters and plasmid constructs could be employed, as described, for example, in U.S. Pat. Nos. 5,276,268 and 5,273,894; in European Patents EP 0,242,236 and EP 0,242,246; and in U.S. patent application Ser. No. 08/113,561.
A xe2x80x9cPAT gene integration event DNA segmentxe2x80x9d is the result of an integration event of a gene encoding a PAT enzyme. This is more particularly defined herein as a DNA sequence encoding PAT as well as adjacent plant DNA sequences, preferably, maize DNA sequences, the function of which flanking sequences are affected by the integration of the gene encoding PAT.
Plants, plant lines or hybrids that contain a PAT gene integration event DNA segment are referred to as GR plants, GR lines, GR inbreds or GR hybrids. In particular, an integration event referred to as the B16 integration event is defined as the DNA sequence derived from pDPG165, including the bar gene, that was introduced into maize through genetic engineering techniques as well as maize DNA sequences, the function of which are affected by the integration of the bar gene in the transformant designated B16.
xe2x80x9cCrossingxe2x80x9d a plant to provide a plant line having an increased yield relative to a starting plant line, as disclosed herein, is defined as the techniques that result in a PAT gene being introduced into a plant line by crossing a starting line with a donor plant line that comprises a PAT gene. To achieve this one would, generally, perform the following steps:
(a) plant seeds of the first (starting line) and second (donor plant line that comprises a PAT gene) parent plants;
(b) grow the seeds of the first and second parent plants into plants that bear flowers;
(c) pollinate the female flower of the first parent plant with the pollen of the second parent plant; and
(d) harvest seeds produced on the parent plant bearing the female flower.
Backcross conversion is herein defined as the process including the steps of:
(a) crossing a plant of a first genotype containing a desired gene, DNA sequence or element to a plant of a second genotype lacking said desired gene, DNA sequence or element;
(b) selecting one or more progeny plant containing the desired gene, DNA sequence or element;
(c) crossing the progeny plant to a plant of the second genotype; and
(d) repeating steps (b) and (c) for the purpose of transferring said desired gene, DNA sequence or element from a plant of a first genotype to a plant of a second genotype.
Introgression of a DNA element into a plant genotype is defined as the result of the process of backcross conversion. A plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid. Similarly a plant genotype lacking said desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.
It is contemplated that an increase in yield in PAT gene transformants may be a result of an effect of PAT gene activity on plant cell metabolism. It is know that phosphinothricin is an analog of the amino acid glutamate. Furthermore, it is known in plants that inorganic nitrogen is first assimilated into the amino acids glutamate and glutamine and subsequently into aspartate and asparagine and that these amino acids served as a nitrogen reservoir for the plant. It is proposed that the activity of the PAT gene may effect nitrogen metabolism in the plant through acetylation of one or more of these amino acids, or perhaps other compounds, and, therefore, PAT gene activity may effect nitrogen metabolism in the plant cell through the assimilation of inorganic nitrogen into organic compounds. It is contemplated that this alteration in nitrogen assimilation may contribute to increased plant vigor and be further manifested in increased yield.
However, irrespective of the underlying mechanism, the present invention provides methods for increasing the yield of a crop plant that are clearly useful. The invention in one embodiment provides a method for increasing yield of a crop plant that comprises stable transformation of a plant with a DNA sequence that encodes the enzyme phosphinothricin acetyltransferase (PAT). In a preferred embodiment the plant is Zea mays. 
In a further embodiment of the present invention, the DNA sequence comprising a PAT gene and adjacent plant genomic DNA sequences is transferred from a PAT gene transformed donor plant to a recipient plant whereby the yield of the recipient plant is increased following introduction of the PAT gene-flanking sequence DNA segment. The DNA sequence is further transferred to other genotypes through the process of backcross conversion and the yield of said backcross converted plants, or hybrids derived therefrom, is increased relative to the unconverted plant.
It is contemplated that increased yield in lines derived by the B16 transformant may be due to the B16 integration event itself. It is proposed that integration of pDP165 DNA sequences in the B16 transformant inactivated a maize genomic DNA sequence that in the untransformed maize plant contributes to a decrease in yield. For example, plasmid DNA sequences may have integrated into and disrupted a gene that encodes a protein that limits yield and in the absence of the expression of said disrupted gene there is an observed increase in yield. Alternatively, the DNA integration event in B16 may have effected the maize genome in such a manner that the activity of a yield enhancing element is increased with a resultant increase in yield. Defining the precise mechanism(s) that operate, although of scientific interest, is not necessary to the practice of the invention.
In another embodiment of the present invention, an integration event of the bar gene in maize, designated B-3-14-7, or alternatively designated B-3-14-4 or E2 or E5 or E2/E5 or B01C16 or B16, increases grain yield when said integration event is introgressed into lines of maize by backcross conversion, including, but not limited to hybrids.
In another embodiment of the present invention, the flanking maize genomic DNA sequences that are situated adjacent to pDPG165 derived DNA sequences in a PAT gene integration event are isolated. Said flanking DNA sequences are employed to identify and clone the maize genomic DNA sequence into which pDPG165 sequences were integrated in the integration event.
In a further embodiment of the present invention, the pDPG165 derived sequences and maize DNA sequences into which pDPG165 was inserted in the integration event are used as a genetic marker in marker assisted breeding for purposes of selecting maize plants with increased yield based on the presence of said pDPG165 or maize DNA sequences into which pDPG165 integrated without having to grow said plants and assay directly for yield. In a preferred embodiment, said integration event is a PAT gene integration event in Zea mays and more particularly is the bar gene integration event in transformant B16.